Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.
In recombinant protein production, the choice of host cell is very important. Many expression systems for recombinant proteins are available and include bacteria, yeast, fungi, insect, plant and mammalian cells. The suitability of each host cell for recombinant protein expression differs significantly and affects both the product formation and subsequent isolation/purification of the final protein produced. Expression systems such as bacteria are incapable of producing specific classes of proteins, which require post-translational modifications such as glycosylation for bioactivity. Furthermore, many therapeutic proteins require complex post-translational modifications such as glycosylation in order to be biologically active.
The use of mammalian expression systems for producing therapeutic recombinant proteins has been well documented. Mammalian cells have the ability to carry out authentic protein folding and complex post-translational modifications, which are necessary for the therapeutic activity of many proteins. As such, many host cell lines have been approved by regulatory bodies for use in the production of therapeutic proteins.
Expression systems such as human cell lines 293 and PER.C6, have been proposed as hosts for the production of recombinant human therapeutic proteins. These cell lines were developed by transforming human embryonic kidney cells (293) and human embryonic retinal cells (PER.C6) with the transforming early region (E1) of adenovirus type 5 (ad5). Since cell lines such as 293 and PER.C6 express the Ad5 E1 region, they are able to complement the growth of defective Ad5 vectors, which have been crippled by deletion of E1. However, a feature of regulatory importance associated with Ad5-transformed cells is their capacity to form tumours in immunodeficient animals such as nude mice. Therefore, the use of Ad5-transformed cells poses a potential oncogenic risk i.e. the risk of transmitting the tumorigenic components of the cell substrate used for recombinant protein to the subject being treated.
Chinese hamster ovary cell lines are routinely used for the production of biopharmaceutical proteins. A number of characteristics make CHO cells very suitable as host cells: high product levels can be reached in CHO cells; they provide a safe production system free of infectious or virus-like particles; they have been characterized extensively; they can grow in suspension to high cell densities in bioreactors, using serum-free culture media; expression systems for gene amplification in CHO cells have been described (DHFR, GS and metallothionein (MT); Page and Sydenham, 1991, Baker et al, 2001 and Bailey et al 2002).
The cell line CHO-K1 has formed the basis for the generation of a variety of CHO cell line derivatives with improved characteristics, such as the Super-CHO cell line (Pak et al 1996). Super-CHO cells were derived from CHO-K1 cells which were genetically engineered to express the genes encoding transferrin and the growth factor, IGF-1.
Polyoma Virus
Polyomaviruses are a family of small DNA tumour viruses comprising the simian virus 40 (SV40) and the mouse polyoma virus.
Polyomaviruses have served as models to study mammalian gene expression, DNA replication and tumorigenesis (Griffin, 1982). The COS-1 cell line supports episomal replication of SV40 origin containing plasmids (Gluzman, 1981) and was developed by transforming CV1 cells with an origin defective mutant of SV40 virus which codes for wild type T antigen. (Gluzman, 1981). Viruses are host specific and can propagate in their permissive host cells. SV40 virus can propagate in monkey cells (CV1). Py is a mouse virus and can propagate in mice and some rodents.
The large T antigen of Polyomavirus is a multifunctional protein having enzymatic activities and the ability to interact with cellular proteins. It can bind DNA and has two nuclear localization signals. It interacts specifically with several pentameric sequences (GAGGC) at the viral origin of DNA replication. The large T antigen also possesses DNA helicase activity. Helicase activity is crucial for viral DNA replication and requires functional ATPase activity and the ability to bind Polyomavirus DNA.
Replication of Polyomavirus DNA within infected cells requires a specific functional viral origin of DNA replication, large T antigen and a set of cellular proteins known as permissive host factors i.e. the only viral protein necessary for DNA replication is the Large T antigen. Viral DNA replication differs from cellular DNA replication in that the viral origin can fire multiple times during S phase, whereas cellular DNA replication is tightly controlled to prevent any region of the genome from being replicated more than once in a single cell cycle.
A study by Heffernan and Dennis in 1991 (Heffernan and Dennis, 1991) successfully demonstrated the use of eukaryotic expression vectors in viral large T antigen-expressing CHO cells. However, transient expression of the desired protein was shown to only last 48-72 hours. The plasmid was not stably maintained within the cell and was quickly degraded or lost when the cell underwent division.
Episomal replication and stable maintenance of the DNA in mouse embryonic stem cells was also examined by Gassmann et al (Gassmann et al 1995), and in U.S. Pat. No. 2002/0146689, which describes the use of Polyomavirus-based plasmids and the dependence of extrachromosomal replication on the expression of large T antigen.
We have observed that Py based expression vectors (plasmids containing Py origin of DNA replication) like SV40 based vectors can replicate several times within the cell and attain high copy number. In contrast to the COS cell expression system where plasmid replication overwhelms and eventually kills the host cell within a few days of transfection, CHO-T cells transfected with Py-origin containing plasmids do not replicate to such an extent. This is presumably due to the semi-permissive nature of the CHO cell for Py DNA replication. In this system plasmid DNA harbouring the Py ori is lost within 3 days of transfection due to degradation and/or cell division and recombinant gene expression is lost. This limits the time to maximize transient protein production to up to 3 days.
Episomal Vectors
In theory, gene therapy is the delivery of corrective genetic material into cells to alleviate the symptoms of a disease or to correct a defective gene. Optimal vectors for gene therapy require 1) high-level and stable expression of the gene of interest, 2) a high transfection efficiency, 3) no integration into chromosomal DNA to avoid effects on the cell's own DNA, and 4) no transformation features that may result in secondary cancers. Vectors meeting all these criteria are not available. Gene transfer by nonviral vector-mediated systems has been shown to be a safe and simple, but relatively inefficient, method for gene delivery. Many of the systems currently available utilize viral vectors derived from retroviruses and adenoviruses. The use of nonintegrating viruses for the construction of an efficient gene-delivery vector has been intensely studied. In particular, EBV-based expression vectors have been considered.
Epstein-Barr Virus
One method of achieving high expression levels without affecting the cellular genome is to use episomal plasmid vectors which replicate extrachromosomally. Episomal vectors, which replicate extrachromosomally in cells over a long period are useful tools for studying the expression of cloned genes and gene therapy, for example, an Epstein-Barr virus (EBV)-derived plasmid.
Epstein-Barr virus (EBV) is a human gamma herpes virus. The viral elements required for both episomal replication and nuclear retention are the cis-acting replication origin (oriP) of the EBV gene and the EBV nuclear antigen-1 (EBNA-1), which interacts with the oriP region. Plasmids containing the oriP and EBNA-1 sequences are maintained as low-copy number DNA episomes in the cell nucleus and replicate once per cell cycle in primate cells. The chromosome-binding activity of EBNA1 secures the separation to the daughter cells during mitosis.
EBV-based vectors are mainly used in primate cells. The failure of EBV-derived vectors to replicate in most rodent cells, including mouse and hamster, forms a serious drawback for gene therapy, since testing EBV vectors in animals, such as mice, and in culture, such as CHO cells, was previously considered to be impossible. In particular, CHO cells do not support EBV viral DNA replication.
Previous studies using EBV-derived vectors in rodent cells have shown that only some rodent cells (C6 and L6) are capable of replicating plasmids containing EBV oriP and encoding EBNA-1 (Mizuguchi, H. Hosono, T., Hayakawa (2000)), and only for a limited amount of time (i.e <5 days). Replication in CHO cells was only possible when large fragments (21 Kb) of undefined human DNA sequences were inserted in the expression vector (Krysan P. J. and Michele Calos. (1993)). Such large plasmid constructs are not amenable to gene transfer studies due to the unstable and undefined nature of such an expression vector, which affects the production of the potentially therapeutic product. Accordingly, there is a need for an expression plasmid, capable of stable episomal replication and maintenance, without containing large fragments of random human DNA.
Episomal vectors have been used to increase the frequency of stable transfection and to achieve reliable transgene expression in many cell types. However, previously described episomal vectors, for example, based on Epstein Barr Virus (EBV) or SV40 large T antigen have demonstrated limitations both in host cell range and maintenance during long term culture (Piechaczek et al (1999); and Heffernan and Dennis (1991)).
Recently, large-scale transient protein production methods have been described for the rapid production of large amounts of recombinant proteins with yields up to 20 mg/L (Durocher et al 2002; Girard et al 2002; Meissner et al 2001; Jordan et al 1998). Such large scale transient expression employs the transformed human embryonic kidney (HEK293) cells and those engineered to express the Epstein-Barr Virus (EBV) nuclear antigen-1 (EBNA-1).
As such, there is a need for an improved mammalian expression system, which can be used for the production of proteins, in particular recombinant therapeutic proteins and can also be useful in human gene therapy models. Since rodent cells (eg. CHO, BHK, NS0) are routinely used to produce recombinant protein therapeutics, it would be beneficial to have an expression system capable of plasmid replication and retention at work in these cells. The ability to quickly produce recombinant proteins early in product development phase in the same host cell type that would likely be employed for the final bioprocess would clearly be advantageous.
There is also a need for an expression system having enhanced transfection efficiencies, which result in greater recombinant protein expression. Furthermore, there is also a need for cloning/expression vectors that are capable of episomal replication and long-term stable episomal maintenance in mammalian cells.
It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.